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Characterization of Microbial Community in the Selected Polish Mineral Soils After Long Term Storage

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Characterization of Microbial Community in the Selected Polish Mineral Soils After Long Term Storage African Journal of Microbiology Research Vol. 7(7), pp. 595-603, 12 February, 2013 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR12.2308 ISSN 1996-0808 ©2013 Academic Journals Full Length Research Paper Characterization of microbial community in the selected Polish mineral soils after long term storage Agnieszka Wolińska*, Zofia Stępniewska and Agnieszka Kuźniar Department of Biochemistry and Environmental Chemistry, Institute of Biotechnology, The John Paul II Catholic University of Lublin, ul. Konstantynów 1 I, 20-708 Lublin, Poland. Accepted 7 February, 2013 The effect of 19-years storage period at air-dried condition (4°C) and impact of soils rewetting on microbial presence were studied. The topsoil (0 to 20 cm) of Mollic Gleysol, Eutric Cambisol, Rendzina Leptosol, Orthic Podzol and Eutric Fluvisol were used in the experiment. It was found that 10-days of soil incubation at full water capacity conditions and room temperature is enough for soil microbial regeneration. The moisture content was determined for a range of water potential (pF) values: 0; 1.5; 2.2; 2.7 and 3.2, corresponding to available water and representing different water availabilities for microorganisms and plant roots. According to the results, soil moisture content significantly increased (P<0.001) the abundance of the total number of bacteria and most probable number (MPN) of ammonia oxidizing bacteria (AOB). Molecular analysis (16S rRNA) shows the dominance of Betaproteobacteria genera with the main representatives of Nitrosomonas, Nitrosospira, Delftia, Comamonas and Pseudomonas, as well as exponent species of Firmicutes genera: Clostridium and Ruminococcus. Key words: 16S rRNA gene analysis, soil water potential, microorganisms abundance, soil rewetting. INTRODUCTION Soil is a complex and dynamic biological system, and still active: not only are they habitat for living organisms, they (in 2013) it is difficult to determine the composition of are formed by these organism and without their presence microbial communities in soil (Nannipieri et al., 2003). We their development is hindered (Havlicek, 2012). should realized that one gram of soil may harbor up to 10 Soil microorganisms must adapt to microhabitats and billion microorganisms of possibly thousands of different live together in consortia with more or less sharp species (Torsvik and Øvreås, 2002; Schloss and boundaries, interacting with each other and with other Handelsmann, 2006). As less than 1% of the soils biota. Bacterial activities have been reported to be bacteria are cultivated under laboratory conditions unevenly distributed in soil, leading to the concept of hot (Schloss and Handelsmann, 2006; Janssen, 2006), soil spots that are linked to local, transient available C for ecosystems are, to a large extent, uncharted. microbial growth and activity (Grundmann et al., 2001; Consequently, soil is a very heterogeneous system that Nannipieri et al., 2003; Frąc and Jezierska-Tys, 2011). comprises a variety of microhabitats with different Hot environments are between the supporting life physicochemical gradients and discontinuous extreme niches that appear to have maintained some environmental conditions (Grundmann et al., 2001; degree of special biotechnological interest (Grundmann Torsvik and Øvreås, 2002; Nannipieri et al., 2003). Each et al., 2001; Tomova et al., 2010). The two main locations soil micelle surrounded by hydrate surface comprises an for active bacteria are believed to be soil pores within individual biotope- the separate environment for surrounding water film, in regions of preferential flow, or microorganisms life. But above all, soils are biologically alternatively entrapped within soil matrix (Grundmann et al., 2001). Analysis of the spatial distribution of bacteria at microhabitat levels demonstrated that more than 80% of the bacteria were located in micropores (2 to 20 μm) of *Corresponding author. E-mail: [email protected]. stable micro-aggregates (Torsvik and Øvreås, 2002; Sey 596 Afr. J. Microbiol. Res. et al., 2008). Such microhabitats offer the most favorable and sediments for up to several hundred million years conditions for microbial growth with respect to water and (Johnson et al., 2007; Hebsgaard and Willerslev, 2009), substrate availabilities, gas diffusion and protection the knowledge on biodiversity in terrestrial conditions is against predation (Torsvik and Øvreås, 2002). Some still scanty. These studies suggest also that nucleic acids results proved that a high diversity of bacteria belonging can persist over geological timescales (DNA sequences to the Acidobacterium division and Prostheobacter were >1 million years old). The long-term survival of bacteria present in small particles, whereas large particles sealed in permafrozen sediments for up to million years harbored microorganisms belonging to the have also recently been investigated (Johnson et al., Alfaproteobacteria (Torsvik and Øvreås, 2002). 2007). The study showed evidence of bacteria surviving Over the past two decades, molecular methods, in samples up to 500,000 years which make this the especially 16S rRNA gene sequencing, have become oldest independently authenticated DNA to date obtained very popular to help identify unknown bacteria (Torsvik from diable cells. Nevertheless, viable microbial cells and Øvreås, 2002; Nannipieri et al., 2003; Janssen, were recovered from Siberian permafrost as old as 3 2006). In turn, this has led to community analysis using million years (Hebsgaard and Willerslev, 2009). total community DNA extracted from the environment. However, currently the investigations concentrated on Polymerase chain reaction (PCR)-based fingerprinting biological life in the soil after its long time of storage techniques give a higher resolution and provide (about 20 years) are strongly limited. Does it mean that information about changes in the whole community there is no biological life in those types of soils? And what structure (Torsvik and Øvreås, 2002). about microbial spores which have potential to be active Life in the soil environment is constantly connected with even after long term of dormancy? Thus, the objectives of drying and rewetting cycles. Liesack et al. (2000) the current study were: (1) to determinate selected indicated that alternate flooding and drainage of the soils groups of the soil microorganisms (AOB, nitrifying and cause spatial and temporal changes of the soil microbial general bacteria) which are activated at first after long communities and processes. Water is a critical resource period storage as a result of soil rewetting, and (2) to and its availability regulates microbial activity within the determine microorganism’s abundance in the different soil matrix, thus periods of water limitation may affect water content conditions. microbial communities through starvation (Gleeson et al., 2008). A study by Jones and Lennon (2010) found that microbial communities are structured by species MATERIALS AND METHODS responses to environmental variables that fluctuate through time. Dormancy is one trait that allows species to Soil description contend with temporal variability of environmental The Institute of Agrophysics of the Polish Academy of Sciences in conditions, and is considered to be a common life history Lublin has a collection of soils from territory of Poland. The five soil strategy among micorbes (Jones and Lennon, 2010). samples used in the current experiment, were taken in 1990 from Rapid rewetting of a dry soil causes microorganisms to the topsoil (0 to 30 cm) of Mollic Gleysol (Kolno, 22°42’E, 52°28’N), undergo osmotic shock, possibly inducing cell lysis and a Eutric Cambisol (Tarnowo, 16°44E, 52°27’N), Rendzina Leptosol release of intracellular solutes (Gleeson et al., 2008; (Bezek, 23°20’E, 50°51’N), Orthic Podzol (Kolnica, 17°20’E, 50°45’N), Eutric Fluvisol (Zawadka, 21°23’E, 49°54’N), and Iovieno and Bååth, 2008). Majority of the anaerobic collected in the Soil Bank resources. Since 1990 till to 2009 (when microbes form spores or resting stages, whereas, the the samples were taken for our investigations), the soils were asporogenous facultative anaerobes modify metabolism stored in darkness, under air-dried conditions in the special, unified to withstand the toxic stress (Das and Dangar, 2008; containers. The temperature was c.a. 4°C and the room was air- Gleeson et al., 2008). Evidently, about 34% anaerobic conditioned. The basic characteristics of the soil samples are bacteria can survive up to 2 years in the dry (toxic) period presented in Table 1. of the flooded soils (Liesack et al., 2000). According to Gleeson et al. (2008), ammonia oxidizing Determination of soils retention abilities bacteria (AOB) and nitrifying bacteria are a microbial functional groups influenced by a variety of environmental Soil samples (five investigated types) were collected using plastic factors, including water content, that dictate community containers (height of 4.5 cm, diameter of 2.9 cm), and pre- parameters, that is, numbers, diversity and activity in situ. incubated at the state of flooding for 10 days at 20°C. After that, they were placed in an airtight chamber, part of a laboratory set Gleeson et al. (2008) noted also that AOB are well LAB o12 (Soil Moisture Equipment Company, USA) before pressure adapted to surviving extreme drought and become active was applied. The instrument for determining water curves is a steel within minutes of rewetting dry soils. These observations pressure chamber, inside of which a porous
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